CN100337549C - Prebiotic and probiotic compositions and method for use in gut-based therapies - Google Patents

Abstract

The present invention provides a microencapsulated and/or enteric-coated composition comprising a mixture of probiotics, prebiotics, and ammonia-philic bacteria with high urease activity, with or without the addition of inhibitors such as creatinine , uric acid, phenol, indole, molecules of medium molecular weight, and inorganic phosphates with specific affinity for uremic toxins, as well as water sorbents. The present invention also provides a method of alleviating the symptoms of uremia in a patient, said method comprising orally administering a microencapsulated and/or enteric-coated composition to a patient suffering from uremia.

Description

Prebiotics and probiotic compositions and their use in the treatment of intestinal disorders

field of invention

The present invention relates to pharmaceutical compositions and methods of using said compositions to treat renal, hepatic and gastrointestinal disorders by eliminating toxins and other metabolic waste products and reducing or inhibiting unwanted bacterial overgrowth. In one embodiment, the pharmaceutical composition comprises a prebiotic, a probiotic bacterium, an ammonophilic bacterium, and an adsorbent, all of which are microencapsulated and/or enteric coated. Alternatively, the prebiotics, probiotics and ammonophilic bacteria are administered aggregated in one microcapsule, while the adsorbent is administered separately in a microencapsulated and/or enteric-coated formulation, if desired. These pharmaceutical compositions are useful for treating kidney and liver diseases and bacterial overgrowth in the gastrointestinal system.

Background of the invention

Kidney disease is the fourth leading disease in the United States, affecting more than 20,000,000 Americans. Each year, more than 90,000 patients die from kidney disease. In recent years, the number of people with chronic renal failure has been increasing at an annual rate of 11%. Each year about 80,000 Americans on dialysis die from various complications and more than 27,000 are on the annual list waiting for a kidney transplant of which only about 11,000 patients receive a kidney transplant. In addition, nearly 250,000 Americans suffer from end-stage renal disease (ESRD), the final stage of chronic kidney failure.

Normally, the waste of nitrogen metabolism in healthy people is mainly excreted in the urine through the kidneys in the form of urea. However, in individuals with kidney disease, as well as many other diseases such as congenital deficiencies of urinary circulating enzymes, waste nitrogen builds up in the body leading to toxic symptoms. Too much ammonium can cause mental retardation and, in severe cases, coma.

Currently, blood or peritoneal dialysis and kidney transplantation are the only treatments. However, the cost of these treatments is very high. For example, in the United States alone in 1996, the annual treatment cost of ESRD exceeded $14 billion. In developing and underdeveloped countries with low health care budgets, ESRD patients cannot use the above-mentioned treatment methods due to the high cost of treatment. Therefore, there is also a need for alternative ways of treating uremia.

Many therapeutic trials have been based on using the intestines to act instead of the kidneys. During normal digestion, the gastrointestinal system delivers nutrients and water into the blood and removes waste and undigested matter through the intestines. The intestinal wall regulates the absorption of nutrients, electrolytes, water, and certain digestive aids such as bile acids. The intestinal wall also acts as a semipermeable membrane, allowing small molecules to pass from the gut into the blood and preventing large molecules from entering the circulation.

Nitrogen waste products such as urea, creatinine, and uric acid, as well as certain other small and medium molecular weight compounds, flow into the small intestine and equilibrate across the intestinal epithelium. Intestinal dialysis studies have shown a daily flux into intestinal fluid of 71 grams of urea, 2.9 grams of creatinine, 2.5 grams of uric acid and 2.0 grams of phosphate (Sparks, R.E. Kidney Int. Suppl. 1975 Suppl 3, 7:373-376). Thus, various invasive and non-invasive attempts have been made to extract uremic waste from the GI tract, including external intestinal fistula, intestinal dialysis, catharsis, oral sorbents and/or capsule urease administration (Twiss, E.E. and Kolff, W.J.JAMA 1951 146:1019-1022; Clark et al., Trans.Am.Soc.Artif.Intrn.Organs 19628:246-251; Pateras et al., Trans.Am.Soc.Artif.Intrn.Organs 196511:292-295; Shimizu et al., Chemical Abstracts 1955103:129004; Kjellstrand et al., Trans. Am. Soc. Artif. Intern. Organs 198127:24-29; and Kolff, W.J. Kidney Int. 1976 10:S211-S214).

Activated charcoal was the first orally administered sorbent to treat uremia. Activated carbon is a highly porous material with a large surface area, which is obtained by carbonizing organic materials such as wood, petroleum, coal, peat and coconut shells and then activating them with steam, carbon dioxide or chemicals such as zinc chloride. Solute adsorption by activated carbon depends on many factors, including the concentration of the solute in the bulk phase, the chemical nature of the solute, temperature, and pH. Generally speaking, activated carbon is more likely to absorb non-polar solutes than polar solutes. In an in vivo experiment, 190 g of activated charcoal was required to remove 450 mg of creatinine (Goldenhersh et al., Kidney Int. 1976 10:8251-8253). This reduced potency is thought to be due to the uptake of other lipophilic compounds such as cholesterol and related bile acids (Kolff, W.J. Kidney Int. 1976 10:8211-8214; Goldenhersh et al., Kidney Int. 1976 10:8251- 8253). Microencapsulated activated carbon has shown that the amount of carbon required can be reduced to 50 grams (Goldenhersh et al., Kidney Int. 1976 10:8251-8253).

AST-120 is a proprietary coating of specially formulated 0.2-0.4mm porous carbon, which has been proven to be a more effective carbon-based adsorbent. It has been published that administering doses of 3.2 to 7.2 g to uremic patients delayed the rise in blood creatinine levels and delayed the onset of renal dysfunction in nephrectomized rats and 27 uremic patients (Owadu, A. and Shiigai, T. . Am. J. Nephrol. 1996 16(2): 124-7; and Okada, K. Takahashi, S. Nephrol. Dial. Transplant. 1995 10(5): 671-6). AST-120 as an oral sorbent also delayed the development of renal failure (Miyazaki, T et al., Nephrol Dial Transplant 2000 Nov; 15(11): 1773-81).

Several studies have shown that intake of dialdehyde-based starch, also referred to as oxidized starch, leads to increased excretion of non-protein nitrogen (Giordano et al., Bull. Soc. Ital. Biol. Sper. 1968 44:2232-2234; Giordano et al., Kidney Int.1976 10: S266-8268; Friedman et al., Proc.Clin.Dia.Trans.Forum 1977 7:183-184). Unlike activated carbon adsorption of uremic solutes is an easily reversible physical process, dialdehyde-based starch Urea and ammonia are bound by chemisorption involving covalently bound dialdehyde groups. However, like activated charcoal, ingestion of large amounts of about 30-50 grams of oxygenated starch removes only 1.5 grams of urea. Other studies of ingestion of both dialdehyde-based starch and activated charcoal demonstrated improvements in the removal of some uremic wastes (Friedman et al., Proc. Clin. Dia. Trans. Forum 19777: 183-184). Furthermore, the adsorption results of dialdehyde-based starches coated with gelatin and albumin were 6 times better compared to uncoated dialdehyde-based starches (Shimizu et al., Chemical Abstracts 198297:222903). Recently, chronic renal failure was shown to develop delayed after administration of chitosan coated with oxidized cellulose (oxycel) or cellulose dialdehyde (Nagano et al., Medline Abstract UI 96058336 1995).

Locust bean gum, a naturally available carbohydrate-based polymeric oral sorbent, administered to uremic patients at a dose of 25 g/day in cottonseed oil has also been shown to remove significant amounts of urea, Creatinine and Phosphate. Furthermore, locust bean gum adsorbs approximately 10 times its own weight in water (Yatzidis et al., Clinical Nephrology 197911: 105-106). Dietary supplementation of fiber gum arabic has also been shown to increase fecal nitrogen excretion and decrease serum nitrogen concentration in chronic renal failure patients on a low-protein diet (Bliss et al., Am.J.Clin.Nutr.1996 63:392-98) .

Encapsulated urease has also been tested as a nonabsorbable oral sorbent to bind ammonia. In a large number of early studies, zirconium phosphate and encapsulated urease were used as a nonabsorbable oral sorbent to bind ammonia (Kjellstrand et al., Trans. Am. Soc. Artif. Intern. Organs 1981 27:24-29) . The use of a liquid membrane capsule device with encapsulated urease to hydrolyze urea to ammonia and citric acid to neutralize ammonia was also investigated (Asher et al., Kidney Int. 1976 10:8254-8258). Soil bacteria have also been used to regenerate urea into metabolically useful amino acids (Setala, K. Kidney Intl Suppl. 1978 8:8194-202).

In addition, genetically engineered cells of E. herbicola have also been encapsulated and shown to convert ammonia into cell-useful amino acids before it is excreted through the gut. Microencapsulated genetically engineered cells of Escherichia coli (E.coli) DH5 have also been shown to be effective in removing urea and ammonia in an in vitro system and in an animal model of uremic rats (Prakash, S. and Chang, T.M.S. Biotechnology and Bioengineering 1995 46:621-26; and Prakash, S. and Chang, T.M.S. Nature Med. 19962:883-887). However, the administration of genetically engineered strains brings control and safety concerns as well as increased ethical concerns that may lead to non-compliance by patients.

In order to effectively treat renal failure, moreover, it has been estimated that at least 10 to 25.0 grams of urea, 1.0 to 2.5 grams of creatinine, and 0.7 to 1.5 grams of uric acid must be removed. Therefore, more effective treatment methods are needed to remove high concentrations of multiple uremic toxins to relieve symptoms of uremic patients.

Small intestinal bacterial overgrowth (SBO) is also another clinical symptom of the development of renal failure in which the glomerular filtration rate drops below 20 ml/min. Nitrogenous waste flows into the small intestine. As a result, the bacterial overgrowth is greater and the ratio of beneficial and pathogenic bacteria is altered, and these pathogenic bacteria can produce secondary toxic substances, such as carcinogenic and mutagenic compounds such as amines, nitrosoamines , phenolic and indole derivatives.

The human gastrointestinal system harbors a complex microbial ecosystem, including a large variety of bacteria. These bacterial groups that colonize the human gastrointestinal tract mainly affect the function of the gastrointestinal tract, thereby affecting human health and well-being. Among these, some bacteria are conditional or considered harmful and can cause adverse conditions such as diarrhea, infection, gastroenteritis and endotoxemia, while some species are considered "probiotics" which contribute to The human organism plays a beneficial role (Holzapfel WH et al., Int J Food Microbiol 1998 May 26;41(2):85-101).

Among these probiotics, bifidobacteria are the most prominent. When Bifidobacterium is alive and viable, it stimulates the immune system and acts competitively to eliminate disease-causing and spoilage bacteria, reduce the amount of ammonia and cholesterol in the blood, and promote the absorption of minerals. In addition, Bifidobacteria have been shown to be protective against colon cancer by reducing the activity of enzymes that convert pro-carcinogens to carcinogens (von Wright et al., Eur J Gastroenterol Hepatol 1999 Nov; 11(11): 1195-1198).

Lactic acid bacteria such as Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum and streptococcus faecium, Streptococcus thermophilus ) are also probiotics. These bacteria have an antagonistic effect on pathogenic microorganisms, stimulate the immune system, improve lactose digestion, have lipolytic activity to make fat more digestible, reduce plasma cholesterol levels, protect the intestinal mucosa to ensure uniform digestion and absorption of nutrients, and produce in some Polysaccharides that are active in tumors, as well as make some microorganisms less viable that produce enzymes that catalyze the conversion of pre-carcinogens to carcinogens.

Probiotics are thought to act in a synergistic manner to reduce and delay the growth of pathogenic/harmful bacteria in the gut (Marteau, PR et al, Am J Clin Nutr Feb; 73(2Suppl): 430S-436S; Cummings JH et al , Am J Clin Nutr 2001 Feb;73(2Suppl):415S-420S).

Gut bacterial flora can be reduced, unbalanced or eliminated in patients receiving antibiotic therapy or other therapies, as well as in individuals with enteritis, kidney disease and liver disease. Furthermore, it has been shown that during normal aging, bifidobacteria populations decrease while concentrations of pathogenic and spoilage bacteria increase (Orrhage K et al., Drugs Exp Clin Res 2000;26(3):95-111) .

The beneficial effects of microorganisms such as bifidobacteria are known to be due in part to their ability to ferment non-digestible sugars present in the colon, commonly known as prebiotics. A prebiotic is a nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon. Prebiotics are generally considered carbohydrates with relatively short chains. Like other carbohydrates such as non-starch polysaccharides, sugar alcohols and resistant starch, prebiotics can reach the cecum and become a substrate for fermentation. However, they differ in their ability to selectively affect the microbiota. To be effective, they must reach the cecum (Bezkorovainy, A. Am J Clin Nutr 2001 Feb;73(2Suppl):399S-405S).

The non-digestible oligosaccharide for optimal prebiotic effect is inulin-type fructan, which is resistant to digestion by stomach acid and pancreatic enzymes in the body. In pure medium, most bifidobacteria are suitable for utilizing these indigestible oligosaccharides. However, many other bacteria can also metabolize them. When inulin and oligofructose or lactulose (Lactulose) were added to the controlled diet, there was a significant increase in bifidobacteria in the colon, and these changes promoted colonic and systemic health by modulating the gut microbiota. Inulin and oligofructose are rapidly and completely fermented by the microflora of the colon, the products of which are acetate and other short-chain fatty acids. The availability of inulin, lactulose and other non-absorbable carbohydrates arises from the fact that they are all disaccharides or oligosaccharides and the human gastrointestinal tract lacks disaccharide enzymes to hydrolyze them. These fermentable carbohydrates then pass into the colon without being digested. In the colon, resident flora with disaccharidases are able to hydrolyze these oligo- or disaccharides and use them as energy for growth. In the process, they produce large quantities of short-chain fatty acids that acidify the gut and, through an osmotic mechanism, draw water into the intestinal lumen, providing a laxative effect, preventing overgrowth, and allowing easy removal of ammonia and other waste nitrogen . Like lactulose, they can also lead to an increase in fecal biomass and, in this case, capture ammonia for bacterial protein synthesis or convert it to ammonium ions. By stimulating bacterial growth and fermentation, prebiotics also affect bowel habits and mild diarrhea (Jenkins, DJ et al., J Nutr 1999 Jul;129(7Suppl):1431S-1433S).

US Patent 5,733,568 teaches methods of using microencapsulated Lactobacillus to treat antibiotic-associated or other acute or chronic diarrhea and skin and vaginal fungal infections. The microencapsulation is said to inhibit the inactivation of the bacilli and deliver them into the gut also avoiding the lactose intolerance seen in diarrhea.

US Patent No. 5,032,399 teaches the use of Lactobacillus acidophilus to adhere to the intestinal mucosa, thereby reducing the side effects of antibiotic therapy on the gastrointestinal tract, which can weaken the beneficial flora.

US Patent No. 5,531,988 teaches the use of immunoglobulins in compositions as a dietary supplement in addition to beneficial bacteria.

US Patent 5,840,318 also teaches a composition of beneficial bacteria capable of modulating an animal's immune system.

The use of probiotics such as Lactobacillus acidophilus has been suggested to reduce bacterial overgrowth and accumulation of uremic toxins and carcinogens. Nonabsorbable carbohydrates in the diet of uremic patients also suggest that probiotics can increase nitrogen excretion. Administration of lactulose and dietary fiber has also been shown to reduce plasma urea by 11-27% and increase fecal nitrogen excretion by 39-62% (Wrong, O., Nature Medicine 2-3, 1997).

However, one of the major problems with these prior art methods described above is that they tend to target individual urotoxic solutes or toxins. Clinically, however, renal, hepatic and gastrointestinal diseases or disorders require relief of a wide variety of symptoms in fact.

Summary of the invention

It is an object of the present invention to provide a complete, gut-based, low-cost alternative treatment for renal insufficiency, hepatic insufficiency, congenital disorders of urea metabolism, and gastrointestinal disorders and diseases. The present invention differs from the prior art in that more than one symptom can be relieved at the same time. Moreover, the beneficial effects of intestinal flora in restoring normal health can be controlled.

Therefore, the present invention provides a pharmaceutical composition containing probiotics, which can restore the normal balance of beneficial and harmful bacteria, remove excess urea waste from normal protein metabolism, thereby reducing the burden on diseased kidneys, and also remove ammonia to avoid Mental retardation and related symptoms. The pharmaceutical composition of the present invention also contains a prebiotic, which can stimulate the beneficial bacteria flora and also ensure the vitality of the probiotics, so that nitrogen sources such as urea and ammonia can be effectively utilized. In addition, the pharmaceutical composition may comprise an aminophilic urea-degrading microorganism having high alkaline pH stability and high urease activity. In some embodiments, probiotics can restore the normal balance between beneficial and harmful bacteria, remove excess urea waste from normal protein metabolism to relieve the burden on diseased kidneys, and remove ammonia to avoid mental retardation and related symptoms, and can be used As ammonia-loving urea-degrading microorganisms.

In one embodiment, the pharmaceutical composition of the present invention may further comprise a water sorbent to remove water during diarrhea and renal insufficiency, and a mixture of sorbents to remove other waste products of nitrogen metabolism and cellular overgrowth Such as uric acid, creatinine, and guanidine, as well as phenols and indoles, and/or inorganic phosphate adsorbents to maintain phosphate balance. In a preferred embodiment, the activated carbon and the inorganic phosphate adsorbent are combined in the composition to create a synergistic effect and to reduce the relative proportion of these two ingredients relative to the probiotics and prebiotics. Moreover, since the probiotics and prebiotics remove most of the urea and impair bacterial overgrowth, the amount of other normal nitrogen waste and bacterial end-products is minimized, so the present application The composition requires less activated carbon and inorganic adsorbents.

In addition, the pharmaceutical composition comprising a probiotic and a prebiotic, and amphiphilic microorganisms can be used prophylactically in patients suffering from acute or chronic symptoms of uremia due to the development of the disease, only when it is necessary to control diarrhea or other gastrointestinal In disorders, water sorbents, sorbent mixtures, and/or inorganic phosphate sorbents may be administered alone.

The pharmaceutical compositions of the present invention are preferably enteric-coated or microencapsulated for delivery to the ileal or colonic region of the intestine of a patient in need thereof.

The pharmaceutical composition is especially useful for preventing or delaying the need for dialysis in renal disease patients, reducing the frequency and duration of dialysis.

Detailed description of the invention

In kidney failure, the glomerular filtration rate decreases and the kidneys cannot maintain blood homeostasis. A homeostatic equilibrium of water, sodium, potassium, calcium and other salts is no longer possible and nitrogen waste cannot be excreted. Water retention leads to edema and, as the concentration of hydrogen ions increases, acidosis. Nitrogen waste builds up, a condition known as uremia, in the blood and tissues. Uretoxins can be defined as solutes that (i) are normally excreted by healthy kidneys, (ii) accumulate to increasing concentrations during the development of renal failure, and (iii) inhibit various physiological and biochemical functions, In conclusion, uremic toxins cause a complex series of clinical symptoms including uremic syndrome. Examples of uremic toxins include, but are not limited to, ammonia, urea, creatinine, phenols, indoles, and medium molecular weight molecules. More specifically, in uremia, serum creatinine concentrations, blood urea nitrogen (BUN), uric acid, and guanidine-based compounds such as N-methylguanidine (NMG) and guanidinosuccinic acid (GSA) all follow the balance of acid groups, Significant changes were produced by abnormalities in electrolyte and water retention. In addition, there are a number of known or unknown small and medium molecular weight substances identified as uremic toxins that can also accumulate. If left untreated, acidosis and uremia can lead to coma and eventually death.

Further, due to the lack of clearance of metabolic wastes, several compensatory and adaptive processes occur, which further complicate the pathology. For example, when renal function is reduced to less than 20% and serum creatinine levels increase to 8 mg/dl, bacterial overgrowth of the normal intestinal flora occurs. Substantially increased normal substrate metabolism and a large number of various toxic amines such as methylamine, dimethylamine, trimethylamine, phenols and indole metabolism also occur in the overgrowth of this bacteria. As small intestinal bacteria grow, so does ammonia release, which then enters the enterohepatic circulation and is converted to urea.

The introduction of renal dialysis has contributed to rapid progress in the clinical management of renal failure and in the interpretation of uremia. A patient with mild renal failure whose serum creatinine level is less than 400 μmol/L does not require kidney replacement therapy such as dialysis or kidney transplantation. However, usually when the serum creatinine level rises to 900 μmol/L, the patient requires routine dialysis or kidney transplantation to maintain life.

For patients with ESRD, dialysis can be used as a life-long therapy. Phosphate binders, such as calcium acetate, calcium carbonate, or aluminum hydroxide, are also commonly used in uremic patients undergoing dialysis to reduce elevated phosphate levels. In general, dialysis is expensive, inconvenient, time-consuming and occasionally produces one or more side effects. If the kidney transplant is successful, the patient can lead a more normal life without the long-term costs. However, the transplant surgery, recovery period, and need for continuous anti-rejection drug therapy still entail high costs. Moreover, suitable donors are often lacking. Alternative methods are therefore needed.

The pharmaceutical composition of the present invention comprises probiotics and a mixture of prebiotics, and ammonia-philic bacteria with high urease activity, and/or adsorbents with specific adsorption affinity for uremic toxins such as creatinine, uric acid , phenols, indoles, medium molecular weight molecules and inorganic phosphates, and water sorbents for the relief of uremia. In a preferred embodiment, the composition includes probiotic bacteria, prebiotics such as inulin, fructan oligosaccharides, lactulose and other plant fibers, and also includes probiotics with high alkaline pH stability and high urease activity. Ammonia urea degrading microorganisms, adsorbents such as locust bean gum with specific adsorption affinity for creatinine and urea, activated charcoal with specific affinity for creatinine, guanidine, phenol, uric acid and undetermined components of medium molecular weight, and water Sorbents such as psyllium fiber, guar gum and locust bean gum.

Preferably the bacterial source of the probiotics is capable of metabolizing urea and ammonia into amino acids which can be utilized, inter alia, by the bacteria or by the patient. Typical bacteria with the above properties are Bifidobacteria and Lactobacilli.

Bacillus pasteurii and Sporosarcinaureae are closely related soil bacteria, both of which have a high affinity for urea. These bacteria are non-pathogenic and harmless. Moreover, they grow well at high concentrations of ammonium ions and alkaline pH, conditions that exist in the intestine, especially in uremic conditions. In Bacillus pasteuriani and Ureasarcinia, ammonium accumulation does not occur and these organisms rely on passive diffusion of ammonia across the cell membrane. Both B. pasteurii and U. sarcini showed low affinity for ammonium with Km values of 55.2 mM and 36.7 mM, respectively. Bacillus pasteuriani and Ureasporium sarcina require high ammonia concentrations (40mM) and highly alkaline pH compared to the pathogen P. vulgaris, which can only grow at neutral pH and low ammonium concentrations of 2mM (Kaltwasser, Morsdorf G. H. Arch Microbiol 1989;152(2):125-31). Furthermore, it is known that the specific activity of urease of Ureasporium sarcina is higher than 9300 μmol urea per minute at pH 7.5.

Thus, examples of bacterial sources useful in the present invention as amphiphilic bacteria include, but are not limited to, Bacillus ureasarcinia, Bacillus pasteurii, domesticated Lactobacillus and Bacillus species, and the new species of Lactobacillus KB-I or other Suitable Bacillus species.

In some embodiments, probiotics can be used to restore a normal balance between good and bad bacteria, remove excess urea waste from normal protein metabolism to relieve the burden on diseased kidneys, remove ammonia to avoid mental retardation and related symptoms, and As ammonia-loving urea-degrading microorganisms. Thus, in this embodiment, no separate ammoniaophilic urea degrading organism is required. Instead, in this embodiment, the probiotic and the ammonia-loving urea-degrading organism comprise bacteria of the same species.

Compositions containing these mixtures are enteric coated and/or microencapsulated. The enteric coating of the composition is specifically designed to deliver the sorbent and bacterial source to the ileal and colonic regions of the intestine where urea solutes and other molecules can occur for maximum absorption. This is preferably achieved by enteric coating materials that disintegrate and dissolve at a pH of 7.5 or higher. Examples of casings having these characteristics include, but are not limited to, zein, polyglycolactic acid, polylactic acid, polylactide-co-glycolide and similar coating materials. Enteric casings also allow the sorbent to be delivered to the area of action in a relatively natural form before it reaches the target area, without binding digestive material to the sorbent.

In addition, the compositions of the present invention are microencapsulated, thus allowing the compositions to function like microscopic dialysis cells as described by Chang, T.M.S. (Artificial Cells, Chapter 5, in "Biomedical Applications of Microencapsulation", pp. Lim, F. ed. CRC Press Florida, pp. 86-100). In this embodiment, the composition is coated with a nonabsorbable polymer that only allows small and medium sized molecules to enter the core where the solvent mixture and bacterial source are located. Examples of non-absorbable polymer coatings for microencapsulation include, but are not limited to, alginate/alginic acid, chitosan, cellulose acetate phthalate, hydroxyethyl cellulose, and the like coating material. Microencapsulation prevents the incorporation of macromolecules and other digestive substances, which substantially impairs the effectiveness of the sorbent to specifically adsorb uremic solutes into the sorbent of the mixture. The microencapsulated capsules passed through the intestine as the sorbent mixture adsorbed multiple urea-toxic solutes, metabolized urea of bacterial origin, ammonia, and urea, from which they were excreted intact. Thus, in this embodiment, since the bacterial source remains within the microcapsules, the patient is protected from the possibility of microbial infection by the bacterial source.

In a preferred embodiment of the present invention, the pharmaceutical composition is both microencapsulated and enteric coated.

The pharmaceutical composition of the present invention may further comprise a phosphate-binding agent such as aluminum hydroxide gel, calcium carbonate or calcium acetate, magnesium hydroxide gel and/or water-binding agents such as psyllium fiber, natural gums such as black locust Bean gum, guar gum or modified starch.

The pharmaceutical composition of the present application is administered orally to a subject in need, thereby slowing the accumulation of toxins and metabolic wastes and/or inhibiting or reducing the overgrowth of unwanted bacteria in the subject. In one embodiment, the pharmaceutical composition is administered to a subject suffering from uremia to alleviate symptoms of uremia. "Amelioration" of uremia means that the composition removes sufficient levels of uremic toxins that the patient suffering from uremia does not require dialysis, or dialysis occurs less frequently, or the duration of dialysis is shortened, or does not require immediate dialysis. The composition of the present invention can also be administered to a subject in need thereof, thereby treating not only renal insufficiency and congenital renal metabolic disorders, but also hepatic insufficiency and gastrointestinal disorders and diseases.

In a preferred embodiment, the composition is administered orally in 2-4 ounces of the emulsion or paste mixed with an easy-to-eat food such as a shake or yogurt or the like. Microencapsulated bacterial probiotics and prebiotics can be administered with a cream or paste sorbent mixture or alone in swallowable gelatin capsules.

Mathematical models of solute transport for oral sorbents have been developed based on controlled diffusion of solutes into the intestinal lumen followed by physical binding or chemical entrapment (Gotch et al., Journal of Dialysis 1976-19771(2):105-144). This model provides a rationale for the removal of solutes from the intestine.

For example, in normal renal function the intestinal clearance of urea is 10-12 ml/min, whereas in patients with severely impaired renal function this value is reduced to 3-4 ml/min. This reduction in clearance is independent of blood urea concentration and is directly related to impaired renal function. Normal creatinine clearance is 2-5ml/min.

Further, at steady state, since the mass production rate is the same as the mass elimination rate, the first-stage sorbent facilitates intestinal clearance of any solute, given by the mass balance equation:

Gs=(Kr+Kg)Cs

where Gs = solute production rate, Kr = renal clearance, Kg = intestinal clearance, and Cs = solute concentration

For a given amount of adsorbent, the adsorption-binding process is saturable. Below the saturation level, since the intestinal clearance of solutes is first order, the above equilibrium can be described as follows:

Cs=Gs/(Kr+Kg).

Above the saturation level, since intestinal clearance is zero, the equation can be written as:

Cs=(Gs-adsorption capacity)/Kr.

These equations are useful for predicting the effectiveness of removing solutes from the intestine based on in vitro studies.

In fact, the model was applied to Friedman's removal of urea data by administering an oral sorbent of oxidized starch to the intestines of uremic patients (Friedman et al., Trans. Am. Soc. Artif. Intern. Organs 1974 20:161-167) . Using these data, the model shows that the maximum adsorption capacity of the native oxidized starch oral sorbent is 1.5 g/day, which is insufficient to replace dialysis or reduce the frequency of dialysis. This model also predicts that for the same patient data with a protein catabolism rate of 0.95 g/kg/24 hr and a urea production rate of 5 mg/min, the maximum adsorption capacity of oxidized starch should be 7.2 urea nitrogen/day and intestinal clearance The rate should be 5.6 mL/min. Adsorption below this value, eg, 5.4 g urea/day, delays monthly dialysis at most, as long as the protein catabolism rate remains at 0.6 g/kg/24 hours. Thus, the model is useful for determining the optimal outcome for different compositions of the composition of the invention to alleviate uremia symptoms in patients.

The following non-limiting examples will provide further illustration of the invention.

Example

Example 1: Sorbent source

Oxidized starch (dialdehyde starch) was purchased from MPD Labs, Feasterville, PA. This is a pharmaceutical grade material (98% pure) with a water content of 13% and a minimum of 90% oxidized material as demonstrated by supplier analysis.

Locust bean gum (minimum purity 99%) was purchased from Sigma-Aldrich, St. Louis, MO.

Activated carbon, Super A, pharmaceutical grade (99.9% pure), was purchased from Norit Corporation of America, GA. Each sorbent was coated with a zein coating, microencapsulated with ethyl cellulose (ethocel), and microencapsulated with ethyl cellulose and then coated with a zein coating as the shell material. Encapsulation is performed using a disc process. Three shell solutions were prepared separately: ethylcellulose only, zein only, zein combined with ethylcellulose. After the shell material was prepared, the various adsorbents were added to the shell solution (in order to provide 60% of the theoretical payload) and mixed to form a dispersion. The dispersion was then sonicated and pumped at approximately 50 grams per minute into a disc rotating at approximately 20,000 rpm to form microspheres. The microspheres were formed inside a heated cone with an internal temperature of nearly 50°C to evaporate ethanol and water. Capsules are collected by cyclone separator. The native particle sizes and the particle sizes after encapsulation with various sorbents are described in Table 1.

Table 1 Oxidized starch Oxidized starch% Urea/creatinine/uric acid bacteria% 0.1 150/30/30 10 0.2 150/30/30 10 0.3 150/30/30 10 0.4 150/30/30 10 0.5 150/30/30 10 Locust Bean Gum Locust Bean Gum % Urea/creatinine/uric acid bacteria% 0.1 150/30/30 10 0.2 150/30/30 10 0.3 150/30/30 10 0.4 150/30/30 10 0.5 150/30/30 10 WATERLOCK ^™ (A-220) WL% Urea/creatinine/uric acid bacteria% 0.1 150/30/30 10 0.2 150/30/30 10 0.3 150/30/30 -10 0.4 150/30/30 10 0.5 150/30/30 10 activated carbon carbon% Urea/creatinine/uric acid bacteria% 0.1 150/30/30 10 0.2 150/30/30 10 0.3 150/30/30 10 0.4 150/30/30 10 0.5 150/30/30 10 single microorganism Urea/creatinine/uric acid Vaccination% 100/15/15 3 100/15/15 5 100/15/15 10 150/30/30 5 150/30/30 10

Scanning electron micrographs (SEM) of raw materials including dialdehyde-based starch, locust bean gum, activated carbon, and aluminum hydroxide, and twelve microcapsule formulations were prepared. Batch 11 produced stick capsules either because they were not fed directly into the middle of the pan or because their solids content was too high. Batch 13 repeated batch 11 but reduced the zein content from 15% to 12%, and it worked.

In addition to scanning electron micrographs of each batch, digital light micrographs were taken.

Example 2: Escherichia coli (E.coli) DHS and other bacterial sources

Following the procedure described by Chang and Prakash (Biotechnology and Bioengineering 1995 46:621-26), genetically engineered E. coli DH5 were inoculated, cultured, grown, harvested and microencapsulated. The probiotics and ammonophilic bacteria of the present invention are also treated in the same way.

Example 3: Formulation

Various probiotics, prebiotics and adsorbents are mixed with different food additives to form different formulations of the present invention. Their general composition is described in Table 2 below.

Table 2: Ingredients and Quantities in a 60 gram Dose (Based on BID Intake) Element Range (grams) The most preferred amount (grams) Probiotics 5-20 12.5 Locust Bean Gum 10-20 15 WATERLOCK ^™ 1-5 3 activated carbon 0.5-2 1 inulin 1-10 5 food additives ~3-~42 ~23 total 60 grams 60 grams

Example 4: In vitro potency evaluation

Prepare simulated gastric juice, which contains NaCl, HCl, and pepsin in distilled water, pH adjusted to 1.2. The formulations were tested for stability at acidic pH and gastric conditions according to USP procedures. Each modified sorbent material and binary and ternary formulations were tested at 37°C for 1-2 hours by stirring approximately 5 grams of the material into a test solution simulating gastric fluid to ensure the integrity of the modified sorbent.

The simulated synthetic intestinal fluid contained potassium dihydrogen phosphate, sodium hydroxide, pancreatin preparation mixture and distilled water, which was also prepared according to the USP test solution preparation and adjusted to pH 7.5. The intestinal fluid was fortified with uremic solutes to obtain a solution containing 150 mg of urea, 30 mg of creatinine and 30 mg of uric acid per 100 ml of synthetic intestinal fluid.

To vary the concentration, the stock solution was diluted with non-fortified intestinal stock solution to obtain 75% and 50% concentrations of variable urotoxic intestinal fluid. First, 100%, 75% and 50% concentrations of uremic intestinal juice were evaluated by 15 g of adsorbent preparations containing 5 g of oxidized starch, locust bean gum and activated carbon, respectively. From these data, various experimental parameters can be optimized including, but not limited to, volume, concentration of uremic intestinal fluid, and timing of pre- and post-treatment. The optimized parameters were then used for all other sorbent evaluations, formulations and experimental determinations. All experimental observations were repeated three times.

However, initially, sorbent/formulation test batches containing 5, 10 or 15 g of sorbent or formulation were treated with 500 ml of fortified intestinal fluid at various concentrations in graduated cylinders and gently shaken for 8 hours. Samples were aspirated from the supernatant at one-hour intervals, centrifuged when necessary, and analyzed for urea, creatinine, and uric acid by commercially available kits (Sigma Diagnostics Company, St. Louis, MO). This cycle can be used once the absorbency and equilibration time have been determined, estimated at least 2 hours.

Lactobacillus sporogenes, Lactobacillus acidophilus, Bacillus pasteuriani (Bp) and Escherichia coli DH5 (Ec) were evaluated by urease activity from enhanced artificial intestinal juice (FAIF: 100/ 150 mg/dL urea, 15/30 mg/dL creatinine and 15/30 mg/dL uric acid in an aqueous pancreatin preparation, KH ₂ PO ₄ and NaOH) to remove urea.

Under sterile conditions, different mixtures of each natural bacteria, activated carbon and locust bean gum were added to 50 ml of FAIF and incubated at 37 °C and 100 rpm. In separate studies, alginate-poly-L-lysine-alginate microcapsules containing B.p. or E.c., approximately 1 mm in diameter, were added in amounts to normalize cellular protein content for 50 ml FAIF . Aliquots were taken at 1, 2, 3, 4, 6, and 24 hours and evaluated for reductions in urea nitrogen, ammonia composition, and uric acid and creatinine stability. All experiments were repeated three times, and the results were averaged.

After the bacterial concentration was optimized, various concentrations of Water Lock( ^R ) (WL) A-220 were added to the 50 mg/dL ammonia solution and the ammonia uptake was evaluated. Analytical determination of the ammonia concentration was determined from the supernatant of the filtered suspension. Various concentrations of locust bean gum (LBG) up to highly concentrated FAIF were then added to the system without the addition of bacteria. After incubation for 2 hours at 37°C and 100 rpm, the system was analyzed by residual creatinine and uric acid. Once the appropriate concentration of LBG is determined, activated charcoal is added in variable amounts to highly concentrated FAIF without the addition of bacteria. The system was incubated at 37°C and 100 rpm, and residual creatinine and uric acid were determined after 2 hours of incubation.

Quantitative analytical methods for urea, ammonia, creatinine, and uric acid were available using commercially available diagnostic kits (Sigma, St. Louis, MO. Cat. Nos. 535 and 171, respectively, and Advanced Diagnostics, Inc. Division of Inamco Group, respectively, South Plainfield, NJ. Cat. Nos. 131500 and CASO-50).

Example 6: TNO Gastrointestinal Model (TIM)

The TNO gastrointestinal model (TIM) closely mimics the successful kinetic conditions in the lumen of the gastrointestinal tract (van der Werf et al., J. Agric. Food Chem. 49, 378-383, 2001). Kinetic parameters simulated include: food and drink intake, pH profile, enzyme and zymogen concentrations in the stomach including saliva and small intestine including pancreatic juice; bile concentration in different parts of the intestine; kinetics of chyme passing through the stomach and small intestine biological pathways; and absorption of water-soluble digestion products and water. In the large intestine model, a complex high-density microflora of human origin ferments undigested food compounds in a natural colonic environment (simulating pH, water uptake, and uptake of microbial metabolites such as short-chain fatty acids and gases). Therefore, this model was used to evaluate the results of different formulations of the composition of the invention in the large and small intestine.

Example 7: In vivo studies

Rats weighing about 150 ± 15 grams, whose chronic renal failure (CRF) degree is similar to human end-stage renal disease, approaching the initial stage of 10 dialysis (10 initiation of dialysis), were tested with oral microencapsulated adsorbent and Effect of Escherichia coli DH5 cells on removing uremic toxins accumulated in CRF and relieving uremic symptoms. Overall, there were 4 groups including the control group, group 1 with chronic renal failure (n=15) and group 2 with acute renal failure. Specifically, male rats weighing 250-300 grams, approximately 8 weeks old, were purchased from Charles River or Harlan, and housed in cages where access to their feces was prohibited. For all animals, baseline measurements of standard clinical chemistry and measurements of 20 compounds considered to be urotoxins were determined. Rats were then subjected to acute or chronic uremia using surgical methods.

Acute renal failure according to Waynforth, H.B. and Flecknell, P.A. Nephrectomy. In "Experimental and Surgical Techniques in Rats" (2nd ed. 1992, Academic Press (Harcourt, Brace, Jovanovich), London, pp. 29, 274-275 ) prepared by the method of bilateral nephrectomy. Postoperatively, rats were randomly paired into age- or size-matched groups and housed in pairs. Rats were fed intragastrically using a curved dosing needle. One group was treated with oral sorbent in conjunction with Kayexylate to control potassium (treatment group). Another group received Kayexylate alone for potassium control (control group). Both groups were closely monitored for at least 7 to 10 days. Efficient sorbents lead to life extension relative to non-treated groups.

Chronic renal failure (CRF) is produced by a two-stage surgical procedure, similar to that described by Niwa et al. The published 5/6 nephrectomy model, but with some modifications was found to cause profound renal failure, similar to the point at which a person approaches the need for hemodialysis. During this modification, a soft plastic box was sutured around the remnant kidney to prevent excessive enlargement of the kidney after contralateral nephrectomy. This also helps control organ bleeding.

Specifically, a two-step approach was used. On the day of the proposed surgical procedure, fast for one hour. Then the animals were anesthetized by inhalation of isoflurane (FORANE). Anesthetized rats were placed ventrally up toward the surgeon from left to right. Both sides were minimally razored and pretreated with povidone-iodine. Prepare a sterile field on the left side of the rat. A dorsoventral incision was made to access the abdominal cavity just below the left ribcage of the rat near the edge of the ribs. The left kidney is freed from its associated tissue and carefully removed, preferably while grasping the perirenal fat. The adrenal gland is loosely attached to the anterior pole of the kidney by connective tissue and fat, the attachments are torn open, and the adrenal gland is carefully removed. The kidney is placed at the point where both poles are tightly connected to the renal artery entering the organ. Loop a 4.0 monofilament noose over the upper pole and tie tight, being careful not to break the nodule. Tie a double knot. Examine organs for excessive blood loss. If necessary, diathermy was used to stop bleeding and oxycel was applied to the cortex. Repeat the same for the lower pole. The remaining kidneys are packed into pre-prepared plastic boxes, preferably SARAN bags, and protected. The organs are then placed back into the abdominal cavity and the incision is closed in two layers. Wipe the area with povidone-iodine and allow the animal to heal. Analgesic buprenorphine (0.25 mg/kg) was administered for pain relief. After about 3-4 weeks, the right kidney was removed. In this procedure, an abdominal approach is used so that the remaining kidney can be examined. Anesthetize the same way, but make a midline incision along the white matter to reduce bleeding. Gently remove the bowel and observe the remaining left kidney to ensure its function. Make some adjustments to the box if necessary. Assuming the left kidney was completely normal, the right kidney was exposed, freed from the capsule (leaving the adrenal gland), and then the renal vessels were bandaged around and the right kidney removed. Place another bandage over the ureter as far away from the kidney as possible, toward the midline, but without damaging or obstructing any surrounding collateral vessels that may be encountered. The bandage is securely tied with a double knot, the blood vessels close to the kidney are blocked, and the kidney is removed. The incision is closed, analgesics are administered to the animal, and the animal is allowed to heal in a warm environment.

With this modified procedure, consistent results were obtained in rats: serum creatinine values of 2.6 ± 0.2 (range 2.3-2.5) and blood urea values of 98 ± 5 with low morbidity, (4 weeks postoperatively, less at 20%).

Approximately 5 days after the second surgery, blood was drawn from the contralateral side to match as closely as possible the values of the rat's body weight, serum creatinine, and blood urea. Divide them randomly into 4 groups. Group A and B form a pair feeding. Group A consisted of CRF rats that did not receive any sorbent, and group B rats received sorbent. The third group, group C, was given adsorbents and allowed to ingest food and water freely. Comparing groups B and C, the effects of uremia on appetite and nutrition were found. The fourth group is the sham operation group D, which is used as a control. Rats were observed for 4-7 months. During this period, blood and urine were collected every two weeks. Using the method described by Dunn et al. in "Analytical Chemistry" 197648: 41-44, dimethyl and/or Trimethylamine was analyzed by gas chromatography. At the same time, body weight was measured weekly, and blood pressure and urine osmalities were measured monthly. Their appetite was measured daily by food and water intake. Periodic urine samples were also collected once a month and spot urine samples were collected every two weeks. At the same time, the skinfold thickness was measured as an auxiliary index to evaluate the nutritional status. Rats were sacrificed at 7 months, and the final blood was drawn for clinical chemistry and other special tests. Blood, urine, and brain tissue were also assayed for dimethylamine. In the above study, the adsorbent in suspension in sterile saline was administered orally to rats using a 12-gauge gastric lavage tube.

Claims (13)

1. A pharmaceutical composition comprising a probiotic, a prebiotic, and an ammonia-loving urea-degrading microorganism with high alkaline pH stability and high urease activity, said composition being selected from the group consisting of alginic acid Salt/alginic acid, chitosan, cellulose acetate phthalate or hydroxyethyl cellulose microencapsulated or material selected from zein, polyglycolactic acid, polylactic acid, polylactide-glycolide The copolymer is enteric coated with a material that allows the composition to be delivered in intact form to its target site and prevents the release of ammonia-philic urea degrading microorganisms. 2. The pharmaceutical composition of claim 1, further comprising a water adsorbent. 3. The pharmaceutical composition of claim 2, wherein the water adsorbent is selected from the group consisting of locust bean gum, psyllium fiber, guar gum and zeolites. 4. The pharmaceutical composition of claim 1, further comprising an inorganic phosphate adsorbent and a specific ureatoxic solute adsorbent other than urea. 5. The pharmaceutical composition of claim 4, wherein the inorganic phosphate adsorbent is selected from aluminum hydroxide gel, calcium hydroxide gel and magnesium hydroxide gel, and the specific ureatoxic solute adsorbent is activated carbon. 6. The pharmaceutical composition of claim 1, wherein the probiotic is selected from Bifidobacterium or Lactobacillus. 7. The pharmaceutical composition of claim 1, wherein the prebiotic is selected from the group consisting of fructan oligosaccharides and arabinan oligosaccharides. 8. The pharmaceutical composition of claim 1, wherein the ammonia-loving bacteria is selected from the group consisting of Bacillus pasteurii, Sporosarcina ureae, Bacillus and Lactobacillus KB-1. 9. The pharmaceutical composition of claim 1, wherein the probiotic bacteria and the ammonia-loving urea-degrading microorganism having high alkaline pH stability and high urease activity comprise the same bacteria. 10. A pharmaceutical composition comprising a probiotic, a prebiotic, an ammonia-loving urea-degrading microorganism with high alkaline pH stability and high urease activity, a water adsorbent, an inorganic phosphoric acid A salt adsorbent and a specific ureatoxic solute adsorbent other than urea, said composition being selected from the group consisting of alginate/alginic acid, chitosan, cellulose acetate phthalate or hydroxyethyl cellulose The material is microencapsulated or enteric coated with a material selected from the group consisting of zein, polyglycolactic acid, polylactic acid, polylactide-co-glycolide which allows delivery of the composition to its target site in intact form and prevents release Ammophilic urea degrading microorganisms. 11. Use of the pharmaceutical composition according to any one of claims 1-10 for the manufacture of a medicament for inhibiting the accumulation of toxins and metabolic wastes and unwanted bacterial overgrowth. 12. The application of the pharmaceutical composition described in any one of claims 1-10 in the preparation of medicines for alleviating the symptoms of uremia. 13. The use as claimed in claim 12, wherein the uremic symptoms are caused by kidney disease or an inborn defect in urea metabolism.